Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

Abstract

Lipopolysaccharide (LPS) is the major glycolipid that is present in the outer membranes (OMs) of most Gram-negative bacteria. LPS molecules are assembled with divalent metal cations in the outer leaflet of the OM to form an impervious layer that prevents toxic compounds from entering the cell. For most Gram-negative bacteria, LPS is essential for growth. In Escherichia coli, eight essential proteins have been identified to function in the proper assembly of LPS following its biosynthesis. This assembly process involves release of LPS from the inner membrane (IM), transport across the periplasm, and insertion into the outer leaflet of the OM. Here, we describe the biochemical characterization of the two-protein complex consisting of LptD and LptE that is responsible for the assembly of LPS at the cell surface. We can overexpress and purify LptD and LptE as a stable complex in a 1∶1 stoichiometry. LptD contains a soluble N-terminal domain and a C-terminal transmembrane domain. LptE stabilizes LptD by interacting strongly with the C-terminal domain of LptD. We also demonstrate that LptE binds LPS specifically and may serve as a substrate recognition site at the OM. T he OM of Gram-negative bacteria is a unique asymmetric lipid bilayer in which the outer leaflet is composed almost entirely of LPS and the inner leaflet of phospholipids (PL) (Fig. 1) (1, 2). Building and maintaining this asymmetric bilayer is a challenge for the cell because the OM is located outside the cytoplasm, in an environment that lacks an obvious energy source such as ATP. LPS and PL are synthesized at the cytoplasmic face of the IM whereas lipoproteins and integral membrane proteins are synthesized in the cytoplasm; all these OM components must be transported across the IM and the aqueous periplasmic compartment to be assembled into the OM (3, 4). In the case of LPS assembly, the molecule must ultimately reach the outer leaflet of the OM (Fig.

The complex that inserts lipopolysaccharide into the bacterial outer membrane forms a two-protein plug-and-barrel

“…Recently, we described the isolation and characterization of LptD/E, the two-protein complex responsible for assembling lipopolysaccharide in the outer membrane of E. coli (10). Here, to develop a more detailed understanding of LptD/E structure, we have carried out in vivo photocrosslinking (13) to map intermolecular interactions within the complex.…”

“…To distinguish between these two possibilities, we compared the stability of purified LptD/E complexes containing wild-type LptD or LptDΔ529-538 in vitro using our previously published methods (10). Like wild-type LptD/E complexes, LptDΔ529-538/E complexes eluted as a single peak from a size-exclusion column and migrated as a single band on nondenaturing blue native (BN-)PAGE (Fig.…”

“…LptE is required for LptD biogenesis (10); proper disulfide bond formation in LptD depends on the presence of LptE (11), but how LptE facilitates this process is unknown. Interestingly, LptE also binds specifically to LPS in vitro, suggesting that it directly handles LPS during transport (10). The mechanism by which LptD/E work together to translocate LPS across the OM and establish the asymmetry of the bilayer is unknown.…”

“…Two of these proteins, LptD and LptE (formerly Imp and RlpB, resp. ), reside in the OM and are responsible for the translocation of LPS across the OM (to the cell surface) in the final stages of assembly (8)(9)(10).…”

Lipopolysaccharide assembly in the bacterial outer membrane revealed by X-ray crystallography

“…Eventually, LPS will be transported across the periplasm and assembled into the bacterial OM, which involves seven essential lipopolysaccharide transporting (Lpt) proteins LptA-LptF [3]. It is believed that the OM-localized LptD/E complex is responsible for the translocation and insertion of LPS into the OM, which is the final step of LPS biogenesis [4].…”